Although the advent of combination antiretroviral therapy (cART) has significantly reduced the severity of the CNS deficits in most patients, the prevalence of milder deficits, collectively referred to as HIV-associated neurocognitive disorders (HAND), remains at pre-CART levels. Increasing evidence suggests that opiate drug abuse, including heroin and morphine abuse, enhances the development and progression of HAND. This is thought to occur via the interaction of opiates with opioid receptor-expressing microglia, astrocytes, and neurons, ultimately leading to a cascade of cellular events that directly/indirectl impacts surrounding neurons. Opiate drugs of abuse chiefly activate the -opioid receptor (MOR) subtype, expressed by various CNS cells. MOR is encoded by the OPRM1 gene, which has been shown to undergo extensive alternative splicing to generate at least 21 splice variants. Neural progenitor cells, which are the undifferentiated precursor cells of CNS neurons and glial cells, are known to be affected by HIV and opiates independently, but we recently showed an interactive effect between HIV and morphine on the proliferation of mouse NPCs and immortalized human NPC (hNPCs), as well as on the populations of cells that they produce. It remains entirely unknown whether similar effects of HIV and morphine occur in primary hNPCs, and if other essential functions of hNPCs are affected. The proposed studies test the hypothesis that HIV and opiates have interactive effects on the survival, proliferation, motility, and differentiation of primary hNPCs that differentially involve MOR splice variants. To support the hypotheses, our preliminary data showed that: 1) HIV-1 and morphine co-exposure significantly decreased the number of hNPCs entering S phase, reduced hNPCs cell density, and significantly increased hNPCs doubling time without affecting hNPC survival; 2) hNPCs express MOR splice variants MOR(1-2) and MOR-1K and their expression was differently modulated by HIV-1. The hypotheses are tested in three specific aims. Aim 1 examines the effects of HIV-1 and morphine co-exposure on essential hNPC functions such as proliferation, differentiation, and motility using multiple biochemical and histochemical assays. Aim 2 tests the functional significance of MOR splice variant signaling in HIV-1 morphine-mediated outcomes by genetic manipulation of expression using siRNAs and over-expression vectors. Resulting phenotypes/behaviors will be examined as described in Aim 1. Aim 3 examines the signaling pathways that are activated by MOR(1-2) versus MOR-1K when NPCs respond to HIV morphine. A combination of functional (e.g. cAMP activation assay) and biochemical (e.g. immunoprecipitation) approaches examine specific signaling through MAP-kinases and PI3K/Akt. Findings from the proposed studies will provide novel information concerning how hNPCs in the CNS are affected by HIV-opiate co-exposure and the involvement of particular MOR splice variants. Our studies may be fundamental to understanding HIV-MOR interactions in other cell types as well, since the separate functional roles of MOR splice variants have not been carefully tested.